Antiviral Activity of Diterpene Esters on Chikungunya Virus and HIV

Article pubs.acs.org/jnp

Antiviral Activity of Diterpene Esters on Chikungunya Virus and HIV Replication Louis-Félix Nothias-Scaglia,†,‡ Christophe Pannecouque,§ Franck Renucci,† Leen Delang,§ Johan Neyts,§ Fanny Roussi,‡ Jean Costa,† Pieter Leyssen,§ Marc Litaudon,*,‡,∥ and Julien Paolini*,†,∥ †

Laboratoire de Chimie de Produits Naturels, UMR CNRS SPE 6134, University of Corsica, 20250, Corte, France Institut de Chimie des Substances Naturelles CNRS-ICSN UPR 2301, University Paris-Sud, LabEx CEBA, 1 Avenue de la Terrasse, 91198, Gif-sur-Yvette, France § Laboratory for Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium ‡

ABSTRACT: Recently, new daphnane, tigliane, and jatrophane diterpenoids have been isolated from various Euphorbiaceae species, of which some have been shown to be potent inhibitors of chikungunya virus (CHIKV) replication. To further explore this type of compound, the antiviral activity of a series of 29 commercially available natural diterpenoids was evaluated. Phorbol-12,13-didecanoate (11) proved to be the most potent inhibitor, with an EC50 value of 6.0 ± 0.9 nM and a selectivity index (SI) of 686, which is in line with the previously reported anti-CHIKV potency for the structurally related 12-O-tetradecanoylphorbol-13-acetate (13). Most of the other compounds exhibited low to moderate activity, including an ingenane-type diterpene ester, compound 28, with an EC50 value of 1.2 ± 0.1 μM and SI = 6.4. Diterpene compounds are known also to inhibit HIV replication, so the antiviral activities of compounds 1−29 were evaluated also against HIV-1 and HIV-2. Tigliane- (4β-hydroxyphorbol analogues 10, 11, 13, 15, 16, and 18) and ingenane-type (27 and 28) diterpene esters were shown to inhibit HIV replication in vitro at the nanomolar level. A Pearson analysis performed with the anti-CHIKV and anti-HIV data sets demonstrated a linear relationship, which supported the hypothesis made that PKC may be an important target in CHIKV replication.

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kinase C isoenzymes).17,18 In particular, phorbol esters activate conventional and novel PKCs by interacting with their cysteinerich C1 domain, triggering a series of interlinking signaling pathways.19 The clinical launch of Picato (ingenol-3-mebutate, PEP005), a broad PKC modulator isolated from E. peplus for the treatment of a precancerous skin condition (actinic keratosis),20 along with recent results on EBC-46 (tiglianetype), which shows promise in curing various tumors by a single intralesional injection in a preclinical model for cancer,21 highlight the therapeutic potential of diterpene esters endowed with PKC modulation abilities. In the present investigation, the antiviral potential of 29 diterpene esters (1−29), belonging to tigliane (1−25), ingenane (26−28), and daphnane (29) types, was evaluated in cell culture against CHIKV, HIV-1, and HIV-2. The aim of this study was to enhance knowledge of the structure−activity relationships (SARs) for these chemical classes and to investigate a possible correlation between their anti-CHIKV and anti-HIV activities.

hikungunya fever is caused by an arthropod-borne virus that is known for causing major epidemics and because of its severe morbidity (inclusive of virus-induced arthralgia, fever, myalgia, and rashes).1 Worldwide travel and introduction of the mosquito vectors Aedes aegypti and A. albopictus are responsible for the spread of chikungunya virus (CHIKV) from Africa and the Indian subcontinent to Southeast Asia, around the Indian Ocean, and more recently to the Caribbean islands and Central and South America.2,3 Currently, no antiviral drugs or vaccines are available for the treatment or prevention of CHIKV infection.4 Recent scientific reviews have highlighted issues and the latest developments in the search for new therapeutic antiviral solutions.5,6 In an effort to identify novel inhibitors of CHIKV replication, Euphorbiaceae species have been selected and investigated by means of bioassay-guided purification, which resulted in the isolation of diterpene esters of daphnane, jatrophane, and tigliane types with anti-CHIKV activity.7−10 Moreover, the structurally related diterpenoids prostratin and 12-O-tetradecanoylphorbol-13-acetate (TPA) were also found to be potent and selective inhibitors of CHIKV replication in vitro.11 TPA and other tigliane-, ingenane-, and daphnane-type diterpenes are known to possess anti-HIV properties, but also pro-inflammatory and tumor-promoting activities,12−16 through the broad activation or other modulation of PKCs (protein © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 26, 2015

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DOI: 10.1021/acs.jnatprod.5b00073 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of diterpene esters 1−29.

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triester 19 (EC50 = 1.7 ± 0.3 μM, SI = 14.2), and to a lesser extent phorbol-13 butyrate (5) (EC50 = 20 ± 10 μM, SI = 12.3) proved to be selective inhibitors of CHIKV replication (SI approximating 10 or above). Most of the other diterpenes (3, 9, 10, 12, 14, 15, 18, 22, 24, 28, and 29) reproducibly inhibited CHIKV-induced cell death, with EC50 values in the μM range, but also caused significant antimetabolic effects, resulting in SIs in the range 1.5 to 6.4. From these results, the following preliminary structure−activity relationship observations could be derived: the length and location of the acyl chain on the 4βOH phorbol diterpene moiety appear to be important determinants for strong anti-CHIKV activity. Considering the phorbol-12 and phorbol-13-monoesters, it was noted that compounds 2 and 4, which possess only one acetyl group at C12 or C-13, were not active, whereas the anti-CHIKV activity was significantly higher with a longer aliphatic side chain

RESULTS AND DISCUSSION The antiviral evaluation of compounds 1−29 (Figure 1) was performed in viral cell-based assays for CHIKV (Indian Ocean strain 899), a member of the genus Alphavirus, and two members of the Lentivirus group, HIV type-1 (strain IIIB) and HIV type-2 (strain ROD) (Table 1). In a previous study,9 using the same methodology, it was established that TPA (13) and prostratin (23) are strong inhibitors of CHIKV replication (EC50 = 2.9 ± 0.3 nM, SI = 1965, and EC50 = 2.7 ± 1.2 μM, SI = 22.8, respectively).11 The results of the present study revealed that phorbol-12,13-didecanoate (11) was the most potent inhibitor evaluated, with an EC50 value of 6.0 ± 0.9 nM, SI = 686, corroborating the strong anti-CHIKV activity previously found for the structurally closely related TPA (13). In addition, phorbol monoesters 6 and 7 (EC50 = 2.2 ± 0.1 μM, SI = 9.7 and EC50 = 0.99 ± 0.03 μM, SI = 9.0, respectively), phorbol B



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Table 1. Antiviral Activities of Diterpene Esters 1−29 against CHIKV, HIV-1, and HIV-2 cpd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 chloroquine (positive control) azidothymidine (positive control)

CHIKVa c

>343 >245 4.9 >174 20 2.2 0.99 9.4 1.8 3.2 6.0 1.5 2.9 2.8 1.1 15 24.6 0.6 1.7 32.6 13.1 0.7 2.7 0.7 50.8 30.1 22.9 1.2 1.8 10 n.d.

SIb

HIV-1 (IIIB)a

c

± 1.7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 0.1 0.03 1.0 0.2 0.2 0.9 nM 0.1 0.3 nMc 0.5c 0.3 4 7.1 0.1 0.3 4.0 0.5 0.1 1.2c 0.1 2.1 19.2 5.2 0.1 0.2 5

>1 0.8 1.5

>343 >307 4.9 >217 122 0.9 0.24 4.8 79 7.0 1.2 >16 0.9 >18 1.5 9.0 >279 36 0.75 27.4 >20 3.3 1.9 0.6 0.3 209 17 27 26 n.d. 7.1

12.3 9.7 9.0 4.2 2.1 1.8 686 2.2 1965c 1.9c 3.3 1.1 1.7 3.7 14.2 2.2 2.7 5.9 22.8c 5.0 1.9 4.8 2.3 6.4 2.3 8.9

± 0.6 ± ± ± ± ± ± ±

18 0.1 0.02 0.8 4 nM 1.0 nM 0.1 nM

± 0.1 nM ± 0.9 nM ± 1.0 nM ± 5 nM ± 0.01 ± 2.9 ± ± ± ± ± ± ± ±

0.4 0.1 0.2 0.4 12 2 nM 7 nM 3

± 0.4 nM

SIb >1 >1 4 1 >3 130 107 >59 >3155 >2886 13 754 n.d. 20 220 n.d. 12 600 12 749 1 483 23 >9 n.d. 5 >171 221 >737 >2 7847 3396 8 >3516

HIV-2 (ROD)a >343 >307 2.0 101 25 0.15 78 1.0 14.0 1.2 0.2 >16 0.7 >17 0.2 3 243 4.0 80.0 6.6 >35 1.2 0.9 0.20 31 131 9 9 22.6 n.d. 6.0

SIb

± ± ± ± ± ±

0.1 nM 2 nM 55 0.1 nM 1.3 nM 2.8

± ± ± ± ± ± ± ±

0.1 0.1 0.02 2 nM 57 1 nM 3 nM 4.2

>1 >1 9 >2 >12 1 1 >274 >17 410 19 162 120 274 n.d. 107 377 n.d. 97 145 28 586 >1 6152 333 >38 n.d. 13 >370 958 >7818 >3 13 451 6049 3

± 0.7 nM

>4176

± ± ± ± ± ± ± ± ±

0.3 12 3 0.02 11 nM 0.2 0.8 nM 0.1 nM 0.2 nM

± 0.3 nM

EC50’s (CHIKV, HIV-1, and HIV-2) are given in μM, unless otherwise stated. Values are the median ± median absolute deviation calculated from at least three independent assays. bSI, selectivity index, calculated as CC50 Vero/(EC50 CHIKV or HIV). n.d. = not determined. cAnti-CHIKV results obtained with the same methodology from Bourjot et al.8

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present (7 > 6 > 5 > 4). In addition, when comparing the antiCHIKV activity of compounds 3 and 6, it can be inferred that the presence of a long-chain ester unit at C-13, rather than at C-12, led to a higher selectivity. As far as the 4β-OH-phorbol12,13-diesters are concerned, a moderate to strong anti-CHIKV activity was observed for compounds possessing long aliphatic side chains at C-12 and C-13 (i.e., 9−11, 13, and 15). Compounds 11 and 13, which possess at least a 10-carbon side chain at C-12, were approximately 1000 times more potent than the other members of the series, stressing the key role of the C-12 acyl side chain and its chain length. As previously shown for TPA (13) and its epimer, 4α-TPA (14),11 comparison of the anti-CHIKV activity of compounds 11 and 12 confirmed that 4β-phorbol derivatives are much more potent against CHIKV than their 4α-counterparts. In addition, since the 20-oxo-phorbol diesters 21 and 22 exhibited a weaker anti-CHIKV effect than their hydroxylated counterparts, 9 and 13, it can be deduced that the presence of a carbonyl group at C-20 has a detrimental effect. Among the 12deoxyphorbol-13-mono- or 13,20-diesters (23−25), prostratin (23) possessed the most favorable anti-CHIKV profile. Finally, the presence of an additional ester group at C-20 did not affect the resultant anti-CHIKV activity, with compounds 18−20 exhibiting moderate to strong activity. For the ingenane-type diterpenes 26−28, ingenol-3,20-dibenzoate 28 was found to

potently inhibit CHIKV replication, whereas the corresponding alcohol 26 and ingenol-3-mebutate (27) showed only moderate activities. Compounds 26−28 are the first examples of ingenane-type diterpenoids to have shown anti-CHIKV activity. In the viral-cell-based assays for HIV (Table 1), phorbol diesters 7, 9−11, 13, 15, and 16, phorbol triesters 18 and 20, and ingenol diterpenes 27 and 28 exhibited strong anti-HIV-1 and anti-HIV-2 activity, with IC50 values in the hundreds of nanomolar to nanomolar range. Moreover, IC50 values were found to be systematically lower for HIV-2. The anti-HIV activities observed for compounds 9−11 and 13 are in good agreement with those reported in the literature.22−25 The selective inhibitory activities against HIV-1 and HIV-2 that were observed for phorbol esters 15, 16, 18, and 19 are reported here for the first time. Compound 16 is a potent PKC activator,26 but has weak tumor-promoting activity when compared to TPA (13).27 Compounds 18 and 19 have been essentially studied for their interaction with the vanilloid receptor.28,29 Overall, the structure−activity relationships that were established for the anti-CHIKV activities of the phorbol, 12deoxyphorbol, and ingenol esters investigated (1−28) were found to be similar to those observed for anti-HIV-1 and antiHIV-2, in terms of the role of the length and the position of the acyl chains at C-12 and C-13, the requirement of a C-4β C



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Figure 2. 3D plot of chemical space of compounds 1−31 using ChemGPS-NP.39,40 Principal component (PC) 1 is linked to size and shape (PC 1); PC 2 to aromatic- and conjugation-related properties; PC 3 to lipophilicity, polarity, and H-bond capacity. The values of selectivity indices (SI) of the most active compounds are specified. Different views are presented in (a) and (b).

surprising given the fact that CHIKV and HIV belong to two different virus genera, the genus Alphavirus and Lentivirus, respectively, but most probably can be explained through a common PKC-based mechanism of action. To expand the interpretation of SARs, the chemical space of diterpene esters 1−29 as well as reference compounds 30 and 31 for CHIKV and HIV replication, respectively, has been generated using ChemGPS-NP.39 This tool performs a principal component analysis (PCA) based on the various physicochemical properties of the different compounds. The results are presented as a 3D plot (Figure 2) using three principal components (PCs) accounting for 82% of the data set variance. These PCs refer to size and shape (PC 1); aromaticand conjugation-related properties (PC 2); and lipophilicity, polarity, and H-bond capacity (PC 3). The results of ChemGPS-NP supported the preliminary SAR observations formulated above. In particular, they illustrated that compounds 11 and 13, which exhibit the most selective anti-CHIKV activity (SI = 686 and 1935, respectively), possess a large molecular size and are relatively nonpolar. However, the results for 12, 14, 15, and 18 (SI < 5) showed that these physical properties are not sufficient to predict anti-CHIKV activity. Furthermore, 5 and 23, which are relatively smaller in size and more polar in comparison with the other diterpene esters evaluated, showed selective anti-CHIKV activities (SI = 12 and 22.8, respectively). A large number of naturally occurring22,41 and chemically modified phorbol and deoxyphorbol esters15,24,42 have been investigated for their potential pro-inflammatory and tumorpromoting activities. In these studies, it has been shown that the C-4 configuration, hydrophobicity, and C-20 functional group are key parameters that modulate these biological activities. Indeed, most of the active compounds share a C-20 hydroxy group and a C-4β configuration and possess long-chain ester moieties at C-12 and C-13.43,44 The mechanism of action by which these compounds act at the molecular level involves modulation of the activity of protein kinase C isozymes17,45

configuration for a strong antiviral effect, and the deleterious effect of the 20-carbonyl group (9 vs 21 and 13 vs 22). Similar observations were made in previous studies.22,23,30 However, in contrast with our previous results on CHIKV, it can be noted that the nature of the acyloxy chain at C-12 does not play such a vital role for the exhibition of a strong anti-HIV effect. Indeed, no loss of potency was observed when a decanoyl unit was replaced by a tigloyl-substituted side chain (11 vs 15) or when a tetradecanoyl moiety was replaced by an N-methylanthranoyl side chain (13 vs 16). When comparing the results of compounds 11 and 18, it is worth pointing out that the presence of a homovanillate ester at C-20 significantly decreased the resultant anti-HIV activities. For the deoxyphorbol derivatives, compounds 24 and 25 showed more potent anti-HIV activity than prostratin (23), which is undergoing human clinical trial development.31 The presence of a 13-phenylacetyl group (25) instead of a 13-isobutyryl group (24) significantly increased the selectivity of the in vitro anti-HIV activity. A previous study has shown that the structurally closely related 12-deoxyphorbol-13-phenylacetate was 20−40-fold more potent than prostratin in inducing latent HIV-1 via PKC modulation.32 Compound 24 was shown to be a broad PKC activator in vivo33,34 but did not show any tumorpromoting activity.26 Also the anti-HIV effects of ingenol esters 27 and 28 were consistent with data previously reported.35−38 Finally, the daphnane orthoester 29 exhibited weak anti-HIV-1 and HIV-2 activity. As a result of the apparent correlation that was observed between the antiviral activities against HIV-1, HIV-2, and CHIKV, a Pearson correlation coefficient was calculated between the EC50 values for each virus pair (Figure 2). The results of this study indicated a strong correlation between the anti-HIV-1 and HIV-2 activities (r = 0.95 ± 0.03) on one hand, but also between the anti-CHIKV and the anti-HIV activities (CHIKV/HIV-1, r = 0.81 ± 0.09; CHIKV/HIV-2, r = 0.84 ± 0.07) on the other hand. At a first glance, these results are D



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through interaction with their cysteine-rich C1 domain.19 PKCs belong to the superfamily of serine threonine kinases that play a central role in intracellular signal transduction implicated in a wide variety of cellular functions.19 It had been shown that an oxygenated functionality at C-20 and the O-acyl function at C13 of tigliane-type diterpenes are essential for their interaction with PKC, as well as their skin-irritant and tumor-promoting bioactivities.16 Nonetheless, the hydrophobicity of phorbol esters appears to be a critical factor that contributes to these biological properties, because it induces different translocation patterns of PKCs in the cell.46−49 In particular, stimulation by the highly hydrophobic TPA (13) leads to translocation of the enzyme to the cell membrane.49 Taking into account that the mechanism of inhibition of HIV replication by phorbol esters,22−24,30,50,51 deoxyphorbol esters,32,52 and ingenol esters35,36 has been reported to be linked to PKC stimulation and that an apparent correlation was found between antiCHIKV and anti-HIV activities, we also postulate that the antiCHIKV activity of diterpene esters might also result from their ability to modulate PKCs. Furthermore, this assumption is supported by similar SARs in both cases. However, some results obtained herein could indicate a different role for PKCs in the anti-CHIKV and anti-HIV activities or different PKC isoforms involved in each virus life cycle. Indeed, 27, a broad potent PKC modulator,53 showed no anti-CHIKV activity, while it exhibited extremely potent activity on HIV replication. In comparison to TPA (13), an another potent PKC modulator, it has been shown that compound 27 stimulated a different pattern of PKC translocation, leading to different biological responses.53 Keeping in mind that 27 elicited no anti-CHIKV activity and the fact that the hydrophobicity of phorbol esters played a preeminent role, the present results may suggested that the pattern of PKC translocation could be an important factor in the mechanism of action of diterpene esters against CHIKV replication.10,54 In conclusion, the current study has allowed the development of preliminary SAR observations among tigliane diterpene esters for CHIKV replication. It has also revealed that ingenane-type esters can exhibit anti-CHIKV activity. Evaluation of compounds 1−29 for selective inhibition of HIV-1 and HIV-2 replication indicated an apparent strong correlation between anti-CHIKV/HIV activities. Furthermore, similar SARs were implicated for inhibition activities of CHIKV and HIV replication, which strengthen the hypothesis of a PKCdependent mechanism. Finally, potent and selective inhibition of HIV-1 and HIV-2 replication exhibited by phorbol esters 15, 16, 18, and 19 and 12-deoxyphorbol-13,20-diesters 24 and 25 is described in this report for the first time.

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triacetate (20), 20-oxo-20-deoxyphorbol-12,13-dibutyrate (21), 12-Otetradecanoyl-20-oxo-20-deoxyphorbol-13-acetate (22), 12-deoxyphorbol-13-acetate (prostratin) (23), 13-O-isobutyryl-12-deoxyphorbol-20-acetate (24), 13-O-phenylacetyl-12-deoxyphorbol-20-acetate (25), ingenol (26), ingenol-3,20-dibenzoate (28), and resiniferatoxin (29). Ingenol-3-mebutate (ingenol-3-angelate) (27) was purchased from Coger SAS (Paris, France). CHIKV Virus-Cell-Based Antiviral Assay. Throughout the experiments, Vero (African green monkey kidney) cells were used. Chikungunya virus (Indian Ocean strain 899), kindly provided by C. Drosten (Institute of Virology, University of Bonn, Germany), was used. Serial dilutions of the test compounds, as well as the reference compound, chloroquine, were prepared in 100 μL of assay medium [MEM Rega3 (cat. no. 19993013; Invitrogen), 2% FCS (Integro), 5 mL of 200 mM L-glutamine, and 5 mL of 7.5% sodium bicarbonate] and added to empty wells of a 96-well microtiter plate (Falcon, BD). Subsequently, 50 μL of a 4× virus dilution in assay medium was added, followed by 50 μL of a cell suspension. This suspension, with a cell density of 25 000 cells/50 μL, was prepared from a Vero cell line subcultured in cell growth medium (MEM Rega3, supplemented with 10% FCS, 5 mL of L-glutamine, and 5 mL of sodium bicarbonate) at a ratio of 1:4 and grown for 7 days in 150 cm2 tissue culture flasks (Techno Plastic Products). The assay plates were returned to the incubator for 6−7 days (37 °C, 5% CO2, 95−99% relative humidity), a time at which maximal virus-induced cell death or cytopathic effect (CPE) is observed in untreated, infected controls. Subsequently, the assay medium was aspirated, replaced with 75 μL of a 5% MTS (Promega) solution in phenol red-free medium, and incubated for 1.5 h. Absorbance was measured at a wavelength of 498 nm (Safire2, Tecan), with the optical densities (OD values) reaching 0.6−0.8 for the untreated, uninfected controls. Raw data were converted to percentages of controls, and the EC50 (50% effective concentration, or concentration calculated to inhibit virus-induced cell death by 50%) and CC50 (50% antimetabolic concentration, or concentration that is calculated to inhibit the overall cell metabolism by 50%) values were derived from the dose−response curves. All assay conditions producing an antiviral effect that exceeded 50% were checked microscopically for signs of a cytopathic effect or adverse effects on the host cell (i.e., altered cell or monolayer morphology). A sample was considered to elicit a selective antiviral effect on virus replication only when, following microscopic quality control, at least at one concentration no CPE or any adverse effect was observed (image resembling untreated, uninfected cells). Multiple, independent experiments were performed. HIV Virus-Cell-Based Antiviral Assay. Evaluation of the antiviral activity of the test compounds against HIV-1 (strain IIIB) and HIV-2 strain (strain ROD) in MT-4 cells was performed using the MTT assay as previously described.55 Azidothymidine was used as positive control. Stock solutions of the test compounds were added in 25 μL volumes to two series of triplicate wells to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman Instruments, Fullerton, CA, USA). Untreated control HIV- and mock-infected cell samples were included for each sample. HIV-1 (IIIB) or HIV-2 (ROD) stock (50 μL) at 100−300 CC/IC50 (50% cell culture infectious dose) or culture medium was added to microtiter tray wells. Mock-infected cells were used to evaluate the effect of test compound on uninfected cells in order to assess the cytotoxicity of the test compound. Exponentially growing MT-4 cells were centrifuged for 5 min at 1000 rpm, and the supernatant was discarded. The MT-4 cells were resuspended at 66 × 105 cells/mL, and a 50 μL volume was transferred to the microtiter tray wells. Five days after infection, the viability of mock- and HIVinfected cells was examined spectrophotometrically by the MTT assay. The MTT assay is based on the reduction of yellow 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Across Organics, Geel, Belgium) by mitochondrial dehydrogenase of metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an

EXPERIMENTAL SECTION

Reference Standards. The purities of the reference compounds were >98%, as determined by HPLC analysis. The following compounds were purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany): phorbol (1), phorbol-12-acetate (2), phorbol12-decanoate (3), phorbol-13-acetate (4), phorbol-13-butyrate (5), phorbol-13-decanoate (6), phorbol-13-tetradecanoate (7), phorbol12,13-diacetate (8), phorbol-12,13-dibutyrate (9), phorbol-12,13dihexanoate (10), phorbol-12,13-didecanoate (11), 4α-phorbol12,13-didecanoate (12), 12-O-tetradecanoylphorbol-13-acetate (13), 12-O-tetradecanoyl-4α-phorbol-13-acetate (4α-TPA) (14), 12-Otiglylphorbol-13-decanoate (15), 12-O-(N-methylanthranilate) phorbol-13-acetate (sapintoxin D) (16), phorbol-13,20-diacetate (17), 12,13-O,O′-dinonanoylphorbol-20-homovanillate (18), 12-O-phenylacetyl-13-O-acetylphorbol-20-homovanillate (19), phorbol-12,13,20E



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eight-channel computer-controlled photometer (Multiscan Ascent Reader, Labsystem, Helsinki, Finland) at two wavelengths (540 and 690 nm). All data have been calculated using the median OD value of three wells. The antiviral experiments were performed in a biosafety screening facility that has been validated for handling of virus as well as the manipulation of molecules of unknown chemical safety risk. All studies have been performed by trained staff. ChemGPS-NP. Chemical space for all structures was calculated using ChemGPS-NP online Web service.40 The 3D plot was generated using the Plot3D package (v. 1.0-2) in R software56 via the RCmdr graphical interface.57 Statistical Analysis. Pearson product-moment correlation coefficients (r) of antiviral activities were computed with the R program56 and using a Pearson product-moment correlation test from the R Commander package.57 This correlation coefficient measures the strength and direction of the linear relationship between two variables, describing the direction and degree to which one variable is linearly related to another.58 The Pearson correlation coefficient can take values ranging from −1 to +1. A value of 1 shows that the variables are perfectly linear related by an increasing relationship, whereas a value of −1 indicates that the variables are perfectly linear related by a decreasing relationship, and a value of 0 shows that no linear correlation exists between the variables. A strong correlation exists if |r| > 0.8 and a weak correlation if 0.5 > |r| > 0.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: + 33 1 69 82 30 85. Fax: + 33 1 69 07 72 47. E-mail: [email protected]. (M. Litaudon). *Tel: + 33 4 95 45 01 97. Fax: + 33 4 95 45 02 57. E-mail: [email protected]. (J. Paolini). Author Contributions ∥

Both senior investigators, M. Litaudon and J. Paolini, contributed equally to the work supervision.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has benefited from an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-25-01). We would like to acknowledge K. Erven, S. Delmotte, C. Collard, N. Verstraeten, and C. Vanderheydt for their excellent technical assistance in the acquisition of the antiviral data. Also, we would like to thank Prof. A. Backlund (Uppsala University, Sweden) for his advice concerning the use of ChemGPS-NP.

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